PROJECT SUMMARY The biliary system stores, modifies, and provides regulated excretion of bile, necessitating exposure to potentially damaging, highly acidic luminal contents. Maintenance of a robust epithelial barrier, limited immune responses to normal bile perturbations, and discrimination between pathogenic and non-pathogenic changes in luminal contents are critical to homeostatic biliary function. Barrier failure or aberrant immune regulation can lead to inflammation, a key component of disease progression in biliary diseases known as cholangiopathies. Despite the critical role biliary quiescence plays in function of the hepatobiliary system, our understanding of homeostatic immune regulation in the biliary tree is limited. One clue may be the striking abundance of tuft cells in the biliary epithelium. Tuft cells, secretory epithelial cells with chemosensory machinery, nucleate an immune cell ?circuit? in the small intestine in which tuft cell IL-25 promotes the production of IL-13 from innate lymphoid cells. Whether similar immune or tissue-modulating roles for tuft cells exist in other tissues is only now being explored. We have found that biliary tuft cells express the core tuft cell gene program, a tissue-specific gene signature, and are sensitive to perturbations in bile acid production. Analysis of gallbladder and extrahepatic ducts from tuft cell deficient mice indicates aberrant immune cell accumulation in this tissue in the absence of tuft cells. The known roles of tuft cells in the small intestine suggests that biliary tuft cells could serve to integrate the biliary epithelium with the immune system; in contrast to the small intestine, our data indicate biliary tuft cells could inhibit inflammatory responses. Together, this suggests that biliary tuft cells could play unappreciated roles in bile acid sensing or the adaptive response to perturbed bile acid abundance, and may regulate biliary immune responses. This proposal will test the hypothesis that biliary tuft cell abundance is controlled by bile content, and that tuft cells repress biliary immune responses at homeostasis. We will take advantage of sensitive, IL-25 based in vivo tools to characterize the abundance, position, and developmental regulation of tuft cells throughout the biliary tree. We will seek to understand the mechanism whereby bile acid manipulation impacts biliary tuft cell abundance using in vitro and in vivo approaches. We will assess how tuft cells might regulate the immune ?tone? of the biliary tree, and what consequences this may have during infection. The secretory and chemosensory functions attributed to tuft cells make these cells attractive integrators of bile content with tissue or immune cell responses, while the unique gene expression profile of biliary tuft cells suggests therapeutic malleability. In addition to the potential for defining novel, tissue-specific roles for biliary tuft cells, our work will add to the existing knowledge on biliary immune responses, as well as the understanding of heterogeneity among biliary epithelial, stromal, and immune cells. Future studies will investigate tuft cell function in more complex models of hepatobiliary inflammation and disease and explore the correlation of tuft cell abundance with human biliary disease states, with great potential to advance new therapeutic targets in biliary pathologies.